Nanoliposomes for sustained delivery of tacrolimus for treatment of anterior segment eye diseases

ABSTRACT

According to the present disclosure, the use of a nanoliposome in the manufacture of a medicament for the prophylaxis and/or treatment of anterior segment ocular diseases is provided. The nanoliposome comprises a plurality of unsaturated and/or saturated lipids forming at least one lipid bilayer encapsulating a hydrophobic drug comprising tacrolimus, wherein the hydrophobic drug and the plurality of unsaturated and/or saturated lipids have a weight ratio of up to 0.2. The present disclosure also provides for such a nanoliposome and a method of preventing and/or treating anterior segment ocular diseases based on the nanoliposomes.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of Singapore PatentApplication No. 10201603094P, filed 19 Apr. 2016, the content of itbeing hereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

The present disclosure generally relates to a nanoliposome comprising ahydrophobic drug such as tacrolimus and the nanoliposome's use in theprevention and/or treatment of anterior segment ocular diseases.

BACKGROUND

Tacrolimus (also known as FK506 or FK-506) is a macrolactum derivativewith immunomodulatory and anti-inflammatory activity. Tacrolimus hasbeen used in eye drops and topical tacrolimus eye drops have beenroutinely used in clinics for management of various anterior segment eyediseases. Generally, the tacrolimus eye drops may be used to treatseasonal allergies (e.g. conjunctivitis), ocular surface diseases (e.g.dry eye), post-operative management of trabeculectomy and in graftrejection. Although prolonged use of the eye drops tend to be necessaryfor better therapeutic response, it may lead to complications such asside effects and patient incompliance. For example, some potentialdrawbacks of using eye drops too often may be sub-optimal management ofthe aforementioned conditions or diseases, and/or serious side effectssuch as permanent redness or damage to blood vessels in the eye.

To mitigate the prolonged use or abuse of the eye drops, an alternativetreatment strategy of providing sustained release of tacrolimus needs tobe developed. The treatment strategy also needs to minimize invasiveand/or risky administration (e.g. intravitreal injection) of tacrolimus.

Based on the above, there is thus a need to provide for a drug deliverymeans which possesses the above advantages while ameliorating one ormore of the drawbacks as mentioned above.

SUMMARY

In one aspect, there is disclosed the use of a nanoliposome in themanufacture of a medicament for the prophylaxis and/or treatment ofanterior segment ocular diseases, wherein the nanoliposome comprises aplurality of unsaturated and/or saturated lipids forming at least onelipid bilayer encapsulating a hydrophobic drug comprising tacrolimus,and wherein the hydrophobic drug and the plurality of unsaturated and/orsaturated lipids have a weight ratio of up to 0.2.

In another aspect, there is disclosed a nanoliposome for use in theprophylaxis and/or treatment of anterior segment ocular diseases,wherein the nanoliposome comprises a plurality of unsaturated and/orsaturated lipids forming at least one lipid bilayer encapsulating ahydrophobic drug comprising tacrolimus, and wherein the hydrophobic drugand the plurality of saturated and/or unsaturated lipids have a weightratio of up to 0.2.

In another aspect, there is disclosed a method of preventing and/ortreating anterior segment ocular diseases by administering ananoliposome comprising a plurality of unsaturated and/or saturatedlipids forming at least one lipid bilayer encapsulating a hydrophobicdrug comprising tacrolimus, wherein the hydrophobic drug and theplurality of saturated and/or unsaturated lipids have a weight ratio ofup to 0.2.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to like partsthroughout the different views. The drawings are not necessarily toscale, emphasis instead generally being placed upon illustrating theprinciples of the invention. In the following description, variousembodiments of the present disclosure are described with reference tothe following drawings, in which:

FIG. 1 shows a cumulative percentage tacrolimus release (%) plot againsttime (days). This plot features an in vitro drug release profile oftacrolimus loaded egg yolk phosphatidylcholine (EggPC) liposomes forthree independent samples (labelled as A, B and C) made according to theembodiments disclosed herein. For each sample, 1 ml of liposomes wasdialysed against 40 ml PBS with a pH of 7.4.

FIG. 2 shows a cumulative percentage tacrolimus release (%) plot againsttime (days). This plot features an in vitro drug release profile oftacrolimus loaded 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC) liposomes for three independent samples made according to theembodiments disclosed herein. For each sample, 1 ml of liposomes wasdialysed against 40 ml PBS with a pH of 7.4. Each data point representsan average reading of the three samples.

FIG. 3 shows the in vitro drug release profile of tacrolimus loaded POPCliposomes for three independent samples. The daily (calculated)tacrolimus mass release (μg) was plotted against time (days). For eachsample, 1 ml of liposomes was dialysed against 40 ml PBS with a pH of7.4. The release profile of tacrolimus based on eye drops is alsoincluded in FIG. 3 as represented by the solid flat line. Each datapoint represents an average reading of the three samples.

FIG. 4 shows a cumulative tacrolimus release (%) plot against time(days). This plot features an in vitro drug release profile oftacrolimus loaded dipalmitoylphosphatidylcholine (DPPC) liposomes forthree independent samples (labelled as E, F and G) made according to theembodiments disclosed herein. For each sample, 1 ml of liposomes wasdialysed against 40 ml PBS with a pH of 7.4.

FIG. 5 shows a non-limiting exemplary embodiment of the nanoliposome asdisclosed herein. Specifically, FIG. 5 shows tacrolimus is encapsulatedin the lipid bilayer.

DETAILED DESCRIPTION

The following detailed description refers to the accompanying drawingsthat show, by way of illustration, specific details and embodiments inwhich the invention may be practiced. These embodiments are described insufficient detail to enable those skilled in the art to practice theinvention. Other embodiments may be utilized and changes may be madewithout departing from the scope of the invention. The variousembodiments are not necessarily mutually exclusive, as some embodimentscan be combined with one or more other embodiments to form newembodiments.

Features that are described in the context of an embodiment maycorrespondingly be applicable to the same or similar features in theother embodiments. Features that are described in the context of anembodiment may correspondingly be applicable to the other embodiments,even if not explicitly described in these other embodiments.Furthermore, additions and/or combinations and/or alternatives asdescribed for a feature in the context of an embodiment maycorrespondingly be applicable to the same or similar feature in theother embodiments.

In the present disclosure, sustained release nanoliposomes encapsulatingtacrolimus have been developed as an effective treatment strategy forvarious anterior segment ocular diseases. Non-limiting examples of suchdiseases may include, inflammatory conditions in the anterior eyesegment (e.g. allergic conjunctivitis), ocular surface problems (e.g.dry eye), and conditions associated with lack of immunosuppression (e.g.post corneal graft). The nanoliposomes of the present disclosure may beapplied via a less risky procedure such as subconjunctival injectioninstead of intravitreal injection.

Advantageously, by using the sustained release nanoliposomes asdisclosed herein, issues associated with existing clinical treatmentstrategies that require the use of tacrolimus eye drops on a daily basisfor prolonged therapeutic efficacy can be circumvented. Not only is theneed to apply eye drops avoided because the nanoliposomes can be appliedvia subconjunctival injection, the drugs are also likely to be releasedover a longer period of time (e.g. days to months) and thus thetherapeutic response can be managed more effectively. This drasticallyimproves patient compliance and minimizes potential side effects arisingfrom frequent eye drops applications required to halt diseases'progression.

With the above in mind, the present disclosure provides for the use of ananoliposome in the manufacture of a medicament for the prophylaxisand/or treatment of anterior segment ocular diseases. The presentdisclosure also provides for such a nanoliposome for use in theprophylaxis and/or treatment of anterior segment ocular diseases. Thepresent disclosure further provides for a method of preventing and/ortreating anterior segment ocular diseases by administering such ananoliposome. Embodiments described in the context of the nanoliposomeand its uses are analogously valid for the method of treating asdescribed herein, and vice versa.

Before going into the details of the nanoliposome and its uses, themethod of treating based on such nanoliposome and the variousembodiments, the definitions of certain terms, expressions or phrasesare first discussed.

In the context of the present disclosure, the term “nanoliposome” refersto a liposome that has a size of at most 200 nm.

In the context of the present disclosure, the term “hydrophobic” refersto materials or substances that are not soluble and/or swellable withwater. Hence, “hydrophobic” materials or substances tend to beimmiscible with water and/or tend to maintain a separate distinct phasefrom water. In this regard, the phrase “immiscible” means that thematerials or substances do not mix and/or tend to separate when left tostand.

The expression of “up to” when used with reference to numerical valuesmay be understood as less than or equal to that numerical value. Inother words, this expression is inclusive of the numerical value itrefers to. For example, “up to 0.2” would cover instances that may beless than 0.2 and instances which are equal 0.2.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

In the context of various embodiments, the articles “a”, “an” and “the”as used with regard to a feature or element include a reference to oneor more of the features or elements.

In the context of various embodiments, the term “about” or“approximately” as applied to a numeric value encompasses the exactvalue and a reasonable variance.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

As used herein, the phrase of the form of “at least one of A and B” mayinclude A or B or both A and B. Correspondingly, the phrase of the formof “at least one of A and B and C”, or including further listed items,may include any and all combinations of one or more of the associatedlisted items.

Unless specified otherwise, the terms “comprising” and “comprise”, andgrammatical variants thereof, are intended to represent “open” or“inclusive” language such that they include recited elements but alsopermit inclusion of additional, unrecited elements. Meanwhile, the terms“consisting” and “consist”, and grammatical variants thereof, areintended to represent “close” or “exclusive” language such that theysolely include the recited elements but exclude additional, unrecitedelements.

Having defined the various terms, expressions and phrases, details ofthe present nanoliposome and its uses, the method of treating based onsuch nanoliposome and the various embodiments are now described below.

In the present disclosure, there is disclosed the use of a nanoliposomein the manufacture of a medicament for the prophylaxis and/or treatmentof anterior segment ocular diseases, wherein the nanoliposome comprisesa plurality of unsaturated and/or saturated lipids forming at least onelipid bilayer encapsulating a hydrophobic drug comprising tacrolimus,and wherein the hydrophobic drug and the plurality of unsaturated and/orsaturated lipids have a weight ratio of up to 0.2.

The hydrophobic drug may be loaded in the nanoliposome up to aconcentration of 1 mg/ml, 0.5 mg/ml, 0.1 mg/ml, or any other loadingswithin these ranges. Hence, the nanoliposome may have a drug loading ofup to 1 mg/ml according to various embodiments. The hydrophobic drugloaded may be a concoction of one or more hydrophobic drugs. In someinstances, the hydrophobic drug loaded may comprise or consist oftacrolimus. The structure of tacrolimus is shown below.

The hydrophobic drug may be encapsulated in the nanoliposome. Thenanoliposome may be formed with at least one lipid bilayer. The lipidbilayer may comprise or consist of a plurality of unsaturated and/orsaturated lipids. Accordingly, the hydrophobic drug(s) may beencapsulated in the lipid bilayer. For instance, tacrolimus may beencapsulated between the hydrophobic tails of the lipids that form thebilayer or any other location within the bilayer.

In various embodiments, the plurality of unsaturated and/or saturatedlipids may be selected from the group consisting of phosphocholines andsphingolipids. In some instances, the unsaturated lipids may comprise orconsist of phosphocholines and/or sphingolipids. In other instances, thesaturated lipids may comprise or consist of phosphocholines and/orsphingolipids.

The phosphocholines may be selected from the group consisting of eggyolk phosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) anddipalmitoylphosphatidylcholine (DPPC). Other suitable phosphocholinesmay also be used to form the at least one bilayer of the nanoliposome.Meanwhile, sphingolipids may include sphingomyelins, sphingosines,ceramides etc.

In preferred embodiments, the plurality of unsaturated and/or saturatedlipids may comprise or consist of at least one unsaturated lipidconstituting more than 50 wt % of the at least one lipid bilayer.

Nanoliposomes which demonstrate better sustained release of hydrophobicdrug(s) (e.g. tacrolimus) over prolonged periods (e.g. several days),may preferably have an unsaturated lipid tail attached to a PC “head” asthe major constituent. The PC “head” may be understood as aphosphatidylcholine (PC) which incorporates choline as a head group.According to preferred embodiments, the at least one unsaturated lipidconstituting more than 50 wt % of the at least one lipid bilayer may becomposed of a phosphatidylcholine (PC) that incorporates choline as ahead group. The structure of a non-limiting exemplary unsaturated lipidhaving a choline group (e.g. POPC) is shown below.

EggPC, as used in various embodiments disclosed herein, may typicallycontain about more than 50 wt % or even about 95 wt % of POPC and/orother PCs with unsaturated tails, along with about 50 wt % or less, oreven about 5 wt % of sphingomyelin which has an unsaturated tail. Thestructures of some major constituents possibly present in EggPC areshown below.

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)

Phosphatidylethanolamine (PE)

Sphingomyelin

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC or di-oleyl)

Dipalmitoylphosphatidylcholine (DPPC)

Notwithstanding the various lipids illustrated, the drug release profileof nanoliposomes consisting of fully saturated lipids may not be ideal.Thus, lipids used to form nanoliposomes may preferably containunsaturated lipid(s) or a mixture of lipids having at least oneunsaturated lipid as the major component. Preferred unsaturated lipidsmay include, but not limited to, POPC, DOPC, sphingolipids etc.Meanwhile, saturated lipids may form the minor components of thenanoliposome. That is to say, saturated lipids in the nanoliposome maybe present at about 50 wt % or less than 50 wt %.

Depending on the type of unsaturated and/or saturated lipids utilized,the nanoliposome may have different sizes. The size of the nanoliposomemay change insignificantly after storage.

In various embodiments, the nanoliposome may have an average size of 80nm to 150 nm, 85 nm to 110 nm, 85 nm to 105 nm, 85 nm to 100 nm, 90 nmto 115 nm, 90 nm to 100 nm, 90 nm to 95 nm, or any other average sizesfalling within these specified ranges. Other sizes may be possibledepending on the lipids used. The nanoliposome may have a size fallingwithin these ranges before storage, with or without drug loaded. Invarious instances, the nanoliposomes may have any of these average sizesbefore storage at 4° C. For example, EggPC nanoliposomes may be 89.64nm±0.78 nm before storage (e.g. storing at 4° C.). In another example,DPPC nanoliposomes may be 106.37 nm±1.00 nm before storage (e.g. storingat 4° C.).

In various other embodiments, the nanoliposome(s) may have an averagesize of 90 nm to 120 nm, 90 nm to 110 nm, 90 nm to 100 nm, 100 nm to 120nm, 110 nm to 120, or any other average sizes falling within thesespecified ranges after storage. Other sizes may be possible depending onthe lipids used. The nanoliposome may have a size falling within theseranges after storage, with or without drug loaded. In various instances,the nanoliposomes may have any of these average sizes after storing(e.g. at 4° C.) for a certain duration (days or months). For example,EggPC nanoliposomes may be 90.67 nm±0.49 nm after storage (e.g. at 4°C.). In another example, DPPC nanoliposomes may be 112.8 nm±1.05 nmafter storage (e.g. at 4° C.).

Based on the change of sizes before and after storage (e.g. at 4° C.),the nanoliposomes may be advantageously stored for extended periods oftime, even with the drug loaded, without having its structural and thedrug integrity compromised. In various instances, the size of ananoliposome may refer to its diameter.

Apart from the size, the type of lipids used may influence thepolydispersity index of the nanoliposome. The polydispersity of thenanoliposome may change insignificantly after storage.

In various embodiments, the nanoliposome may have a polydispersity indexof less than 0.3, 0.2 or even 0.1 before and after storage. Otherpolydispersity index may be possible based on the type of lipids used.In some instances, EggPC nanoliposomes may have a polydispersity indexof 0.052±0.018 before storage while DPPC nanoliposomes may have apolydispersity index of 0.28±0.016 before storage. After storage (e.g.at 4° C.), the EggPC nanoliposomes may have a polydispersity index of0.014±0.007 while the DPPC nanoliposomes may have a polydispersity indexof 0.218±0.036 according to various instances. Based on thepolydispersity index before and after storage (e.g. at 4° C.), thisagain demonstrates the present nanoliposomes may be stored for extendedperiods of time, even with the drug loaded, without having the integrityof the nanoliposome and the drug compromised.

According to various embodiments, the nanoliposome as described abovemay be a multilamellar vesicle or an unilamellar vesicle.

As mentioned above, the present disclosure also relates to ananoliposome for use in the prophylaxis and/or treatment of anteriorsegment ocular diseases, wherein the nanoliposome comprises a pluralityof unsaturated and/or saturated lipids forming at least one lipidbilayer each encapsulating a hydrophobic drug comprising tacrolimus, andwherein the hydrophobic drug and the plurality of saturated and/orunsaturated lipids have a weight ratio of up to 0.2. Embodimentsrelating to the nanoliposome have been described above and arereiterated below.

The nanoliposome may have a drug loading of up to 1 mg/ml, 0.5 mg/ml,0.1 mg/ml, or any other loadings within these ranges. The hydrophobicdrug may be a concoction of one or more hydrophobic drugs. Thehydrophobic drug may comprise or consist of tacrolimus.

The hydrophobic drug may be encapsulated in the nanoliposome, whereinthe nanoliposome may have at least one lipid bilayer formed from theplurality of unsaturated and/or saturated lipids. Accordingly, thehydrophobic drug(s) may be encapsulated in the lipid bilayer. Forinstance, tacrolimus may be encapsulated between the hydrophobic tailsof the lipids that form the bilayer or any other location within thebilayer.

The plurality of unsaturated and/or saturated lipids may be selectedfrom the group consisting of phosphocholines and sphingolipids. Thephosphocholines may be selected from the group consisting of egg yolkphosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) anddipalmitoylphosphatidylcholine (DPPC). The constituents of EggPC havebeen described above. Other suitable phosphocholines may be used to formthe at least one bilayer of the nanoliposome. Meanwhile, sphingolipidsmay include sphingomyelins, sphingosines, ceramides etc.

In preferred embodiments, the plurality of unsaturated and/or saturatedlipids may comprise or consist of at least one unsaturated lipidconstituting more than 50 wt % of the at least one lipid bilayer.

Preferred embodiments of nanoliposomes which demonstrate bettersustained release of hydrophobic drug(s) (e.g. tacrolimus) overprolonged periods (e.g. several days) may comprise or consist of anunsaturated lipid tail attached to a PC “head” as the major constituent.This means that the at least one unsaturated lipid constituting morethan 50 wt % of the at least one lipid bilayer may be composed of aphosphatidylcholine (PC) that incorporates choline as a head group.

Depending on the type of unsaturated or saturated lipids utilized, thenanoliposome may have different sizes before or after storage. The sizeof the nanoliposome may change insignificantly after storage.

The nanoliposome may have an average size of 80 nm to 150 nm, 85 nm to110 nm before storage (e.g. at 4° C.). Embodiments concerning othersizes or range of sizes before storage have been described above.

The nanoliposome may have an average size of 90 nm to 120 nm afterstorage (e.g. at 4° C.). Embodiments concerning other sizes or range ofsizes after storage (e.g. at 4° C.) have been described above.

As explained above, the type of lipids used may influence thepolydispersity index of the nanoliposome. The polydispersity of thenanoliposome may change insignificantly after storage. In variousembodiments, the nanoliposome may have a polydispersity index of lessthan 0.3, 0.2 or even 0.1 before and after storage. Non-limitingembodiments based on EggPC nanoliposomes and DPPC nanoliposomes beforeand after storage have been described above.

The nanoliposome may be a multilamellar vesicle or an unilamellarvesicle according to various embodiments disclosed herein.

As mentioned above, the present disclosure further relates to a methodof preventing and/or treating anterior segment ocular diseases byadministering a nanoliposome comprising a plurality of unsaturatedand/or saturated lipids forming at least one lipid bilayer encapsulatinga hydrophobic drug comprising tacrolimus, wherein the hydrophobic drugand the plurality of saturated and/or unsaturated lipids have a weightratio of up to 0.2.

The present method is advantageous as it mitigates the risk ofintravitreal injection as the present nanoliposome may be administeredby a less risky procedure of subconjunctival injection. Advantageously,subconjunctival injection is able to sustain the release of thehydrophobic drugs from the present nanoliposome which then circumventsthe need for frequent topical administration of tacrolimus eye drop(s)and thus avoiding the side effects of frequent topical administration ofeye drop(s). Embodiments as described above regarding the nanoliposomeand its uses are applicable for the present method of treating anteriorsegment ocular diseases.

In various embodiments, the nanoliposome administered may have a drugloading of up to 1 mg/ml, 0.5 mg/ml, 0.1 mg/ml, or any other loadingswithin these ranges. The hydrophobic drug may be a concoction of one ormore hydrophobic drugs. The hydrophobic drug may comprise or consist oftacrolimus.

The hydrophobic drug may be encapsulated in the nanoliposome, whereinthe nanoliposome has at least one lipid bilayer of the nanoliposomewhere the hydrophobic drug (e.g. tacrolimus) is encapsulated asdescribed above.

The plurality of unsaturated and/or saturated lipids of the nanoliposomeadministered may be selected from the group consisting ofphosphocholines and sphingolipids. The phosphocholines may be selectedfrom the group consisting of egg yolk phosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) anddipalmitoylphosphatidylcholine (DPPC). The constituents of EggPC havebeen described above. Other suitable phosphocholines may be used to formthe at least one lipid bilayer of the nanoliposome. Meanwhile,sphingolipids may include sphingomyelins, sphingosines, ceramides etc.

In preferred embodiments, the plurality of unsaturated and/or saturatedlipids may comprise or consist of at least one unsaturated lipidconstituting more than 50 wt % of the at least one lipid bilayer.

As disclosed above, nanoliposomes of preferred embodiments demonstratebetter sustained release of hydrophobic drug(s) (e.g. tacrolimus) overprolonged periods (e.g. several days) when they possess an unsaturatedlipid tail attached to a PC “head” as the major constituent. In otherwords, the at least one unsaturated lipid constituting more than 50 wt %of the at least one lipid bilayer may be composed of aphosphatidylcholine (PC) that incorporates choline as a head group.

Depending on the type of unsaturated or saturated lipids utilized, thenanoliposome administered may have different sizes before or afterstorage. The size of the nanoliposome may change insignificantly afterstorage.

The nanoliposome administered may have an average size of 80 nm to 150nm, 85 nm to 110 nm before storage (e.g. storing at 4° C.). Embodimentsconcerning other sizes or range of sizes before storage have beendescribed above. Non-limiting embodiments based on EggPC and DPPCliposomes are also described above.

The nanoliposome administered may have an average size of 90 nm to 120nm after storage (e.g. at 4° C.). Embodiments concerning other sizes orrange of sizes after storage (e.g. at 4° C.) have been described above.Non-limiting embodiments based on EggPC and DPPC liposomes are alsodescribed above.

As explained above, the type of lipids used may influence thepolydispersity index of the nanoliposome to be administered. Thepolydispersity of the nanoliposome may change insignificantly afterstorage. In various embodiments, the nanoliposome administered may havea polydispersity index of less than 0.3, 0.2 or even 0.1 before andafter storage. Non-limiting embodiments based on EggPC nanoliposomes andDPPC nanoliposomes before and after storage have been described above.

The nanoliposome administered may be a multilamellar vesicle or anunilamellar vesicle according to various embodiments as described above.

In all the embodiments described herein, the anterior segment oculardiseases may refer to ocular inflammatory diseases, ocular surfacediseases, and diseases or disorders associated with lack ofimmunosuppression (e.g. ocular graft rejection).

In summary, the present disclosure relates to nanoliposomes for use inthe delivery of tacrolimus to treat various inflammatory diseases ordisorders of the eye, ocular surface diseases and in immunosuppressionfor preventing ocular graft rejection (e.g. post corneal graft).

The nanoliposomes may be prepared from lipids such as, but are notlimited to, phosphocholines and sphingolipids. More specifically, thenanoliposomes containing tacrolimus in desired loading concentrationsmay be prepared by passive loading techniques.

Advantageously, the drug loaded nanoliposomes may be applied viasubconjunctival injections and may sustain the release of drugs over anextended duration (days to months). This drastically improves patientcompliance as well as minimizes side effects associated with frequenttopical instillations (e.g. eye drops) to halt the progression ofdiseases.

The nanoliposomes may be referred to as a composition of liposomalmatter that comprises or consists of saturated and/or unsaturatedlipids. The encapsulated drug loading in each of the liposomes may be upto 1 mg/ml and the drug/lipid weight ratio may be up to 0.2. Thiscomposition advantageously sustains the release of tacrolimus beyond twoweeks.

While the methods described above are illustrated and described as aseries of steps or events, it will be appreciated that any ordering ofsuch steps or events are not to be interpreted in a limiting sense. Forexample, some steps may occur in different orders and/or concurrentlywith other steps or events apart from those illustrated and/or describedherein. In addition, not all illustrated steps may be required toimplement one or more aspects or embodiments described herein. Also, oneor more of the steps depicted herein may be carried out in one or moreseparate acts and/or phases.

EXAMPLES

The present invention relates to nanoliposomes for sustained deliveryand/or release of tacrolimus for treatment of ocular diseases. Based onthe examples described below, high loading concentrations of tacrolimusin nanoliposomes were achieved using a passive loading technique. Inaddition, controlled and sustained release of tacrolimus from thenanoliposomes were achieved in an in vitro dialysis. The examplesdemonstrated the release of drug was sustained beyond two weeks based onthe in vitro dialysis.

Further, the examples below exemplify encapsulation of tacrolimus inhigh loading concentrations in the nanoliposomes, where a desirably highdrug/lipid (D/L) weight ratio of up to 0.2 is achieved for bothunstaturated and saturated liposomes. When the D/L ratio exceeds 0.2,the encapsulation efficiency for the drug decreases, thereby resultingin the lost of drugs from the nanoliposomes. An example of howtacrolimus is encapsulated in the nanoliposome is shown in FIG. 5.

Example 1: Preparation of Tacrolimus Loaded Liposomes

Lipids were weighed and placed in a vacuum desiccator for 1 hour toremove any residual moisture before weighing. The drug, tacrolimus, wasalso desiccated before weighing. Each batch of liposome was prepared in5 ml, with an initial lipid concentration of 18 millimolar (mM).

The weighed lipids and drug were then mixed in a round bottom flask, anddissolved in an organic phase mixture that contained methanol andchloroform in a ratio of 1:2. The flask was then maintained in a waterbath temperature of 40° C. and rotated in a rotary operator underreduced pressure for 1 hour to remove the organic phase, ultimatelyleaving behind a thin drug loaded lipid film covering the bottom of theflask. To the thin lipid film, 5 ml of phosphate buffer solution (PBS ofpH 7.4) was added to instantaneously form drug loaded multilamellarvesicles (MLVs).

The liposomes' (MLVs) sizes were reduced by extrusion throughpolycarbonate filter membranes in the corresponding size sequence of 0.2μm (5 times), and 0.08 μm (10 times) using a bench top extruderpurchased from Northern Lipids Inc, Canada. After these extrusion steps,the drug loaded large unilamellar vesicles (LUVs) with a sizedistribution of about 100±20 nm were formed.

Example 2: Partition Coefficient Measurements

Hydrophobic drugs, such as tacrolimus, distribute between the lipidbilayer and continuous phase of the buffer based on the drugs'solubility. Partition coefficient was calculated based on the ratio ofconcentrations measured between these two phases. Partition coefficientvalues were estimated from MLVs prior to the extrusion step.

Briefly, MLVs in microfuge tubes were centrifuged at 13000 rotation perminute (rpm) or 16249 relative centrifugal force (rcf) for half an hour.A clear separation of the lipid pellet from the supernatant was possibledue to the micron sized vesicles. The supernatant was visually clear andthis showed a complete separation was possible.

The total amount of drug(s) in the MLVs and the amount of drug(s) in thesupernatant were estimated by high performance liquid chromatography(HPLC). A continuous (buffer) phase drug(s) amount was calculated fromthe drug measured in the supernatant. The amount of drug partitionedinto the bilayer is calculated by subtracting the drug measured in thesupernatant from the total drug amount. The drug partition coefficient(P.C.) in MLVs was then estimated using the following expression givenbelow.

${P.C.} = \frac{{{Total}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {drug}} - {{amount}\mspace{14mu} {of}\mspace{14mu} {drug}\mspace{14mu} {in}\mspace{14mu} {buffer}}}{{Amount}\mspace{14mu} {of}\mspace{14mu} {drug}\mspace{14mu} {in}\mspace{14mu} {buffer}}$

Example 3: Measurement of Entrapped Drug Concentration

Generally, the various liposome samples were broken with isopropylalcohol (IPA) in a volume ratio of 1:5 (liposome:IPA) and then dilutedwith PBS of pH 7.4. The HPLC method was used to measure the drugconcentration of liposomes while taking into consideration the dilutionfactor and comparing against the standard of tacrolimus in PBS of pH7.4.

Example 4: In Vitro Drug Release Study and Actual Drug Release Per Day

A dialysis method was used to evaluate release of tacrolimus fromliposomal nanocarriers. In the examples, the receptor medium wasphysically separated from the drug loaded liposomes by a dialysismembrane. The released drug concentration was evaluated from thereceptor medium over time by HPLC. For this example, 1 ml of the drugloaded liposomes were filled into a cellulose ester dialysis bag (with amolecular weight cut-off (MWCO) of 100 kD) and clipped by dialysis clipson both ends. 40 ml of PBS buffer at a pH of 7.4 (137 mM NaCl, 2.68 mMKCl, 1.76 mM KH₂PO₄, 10.14 mM Na₂HPO₄) was measured and taken in anamber bottle. The dialysis bags was suspended in the PBS buffer andplaced in an incubator at a temperature of 37° C. 2 ml of dialysismedium was sampled out every day and the entire PBS medium wasreplenished daily. The amount of drug(s) released over time was measuredusing HPLC and presented as a cumulative percentage release plottedagainst time.

Example 5: Characterization of Drug Loaded Liposomes—Size StabilityStudies

The average size as well as the size distribution (polydispersity index)of the liposomes were characterized by using Malvern Zetasizer Nano ZS.The particle sizes were measured after preparation and continuouslymonitored during storage (4° C.).

Example 6a: Results—In Vitro Size Stability of Tacrolimus in UnsaturatedLiposomes (EggPC Liposomes)

The unsaturated liposomes were based on egg yolk phosphatidylcholine(EggPC) liposomes. EggPC contains, without being limited to, POPC,phosphatidylethanolamine, DPPC, DOPC and sphingomyelins etc. The EggPCliposomes have a D/L weight ratio of 0.2 with a drug (tacrolimus)loading concentration of 1 mg/ml. The loading concentration may dependon what is required to adequately deliver daily amounts over severaldays.

The changes in the size of the liposomes upon and during storage werecontinuously monitored with Zetasizer (Malvern Instruments, Malvern,UK). As shown in Table 1 below, tacrolimus loaded EggPC liposomes werefound to be stable for at least one month in storage at 4° C. and theaverage size (Z_(avg)) of the liposomes were found to be about 90 nmwith a narrow polydispersity index (PDI) of less than 0.1.

TABLE 1 Size Measurements of Tacrolimus Loaded EggPC LiposomesImmediately After Extrusion And One Month After Storage At 4° C.EggPC/Tacrolimus LUV D/L weight ratio of 0.2 Initial Drug Concentrationof 1 mg/ml Days Z_(avg) - Zeta size (average) (nm) PDI 0 89.64 ± 0.780.052 ± 0.018 30 90.67 ± 0.49 0.014 ± 0.007

Example 6b: Results—In Vitro Drug Release Study of Tacrolimus inUnsaturated Liposomes (EggPC Liposomes)

The release of tacrolimus from EggPC liposomes was evaluated by adialysis technique and expressed in terms of cumulative drug release (%)over time as shown in FIG. 1. In FIG. 1, the in vitro drug releasestudies (1 ml of liposomes dialysed against 40 ml of PBS with pH of 7.4)for three independent samples of tacrolimus loaded EggPC liposomes ascarried out are shown. All three samples were of the same drug loadingand D/L ratio.

The release of tacrolimus from the liposomes was sustained beyond twoweeks in vitro (approximately 60%), albeit a burst release was observedin the first few days.

Example 6c: Results—In Vitro Drug Release Study of Tacrolimus inUnsaturated Liposomes (POPC Liposomes)

The unsaturated liposomes in this example were based on1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The release oftacrolimus from these liposomes was evaluated by the dialysis techniqueas described above and expressed in terms of cumulative drug release (%)over time as shown in FIG. 2. In FIG. 2, the in vitro drug releasestudies for three independent samples of tacrolimus loaded POPCliposomes (1 ml of liposomes dialysed against 40 ml of PBS with pH of7.4) as carried out are shown. The cumulative percentage of tacrolimusrelease was then plotted against time (days). The almost-completerelease of tacrolimus from POPC was sustained for 42 days in vitro witha suppressed burst observed in the first few days. The drug loadingconcentrations and D/L ratios for POPC liposomes are the same as EggPC.

A comparison of tacrolimus release profiles between tacrolimus eyedrop(s) and tacrolimus POPC liposome is shown in FIG. 3. In FIG. 3, thein vitro drug release studies for three independent samples oftacrolimus loaded POPC liposomes (1 ml of liposomes dialysed against 40ml of PBS with pH 7.4) as carried out are shown. Daily (calculated)tacrolimus mass release profile was plotted against time (days) as shownin FIG. 3. The release profile of tacrolimus based on eye drops is alsoincluded in FIG. 3 as represented by the solid flat line.

Compared to conventional regimen(s) for tacrolimus eye drops where adaily release rate of 0.72 μg/day is needed (represented as a solid flatline in FIG. 3), it is observable that in vitro tacrolimus release fromliposomes of the present disclosure meets or even exceeds theconventional release target for the entire duration of sustainedrelease. Based on these results, the present liposomes not onlycircumvent the issues associated with tacrolimus eye drops but alsoprovide an improved duration of sustained drug delivery.

Example 7a: Results—In Vitro Size Stability of Tacrolimus in SaturatedLiposomes (DPPC Liposomes)

The saturated liposomes were based on dipalmitoylphosphatidylcholine(DPPC) liposomes prepared according to example 1. The DPPC liposomeshave a D/L weight ratio of 0.2 with a drug (tacrolimus) loadingconcentration of 1 mg/ml. As shown in Table 2 below, drug loaded DPPCliposomes were stable in size for at least 1 month storage at 4° C.

TABLE 2 Size Measurements of Tacrolimus Loaded DPPC LiposomesImmediately After Extrusion And One Month After Storage At 4° C.DPPC/Tacrolimus LUV D/L weight ratio of 0.2 Initial Drug Concentrationof 1 mg/ml Days Z_(avg) - Zeta size (average) (nm) PDI 0 106.37 ± 1.00 0.28 ± 0.016 30  112.8 ± 1.05 0.218 ± 0.036

From Table 2, the average size (Z_(avg)) of the liposomes were found tobe about 110 nm with a narrow polydispersity index (PDI) of less than0.3.

Example 7b: Results—In Vitro Drug Release Study of Tacrolimus inSaturated Liposomes (DPPC Liposomes)

The release of tacrolimus from DPPC liposomes was evaluated by thedialysis technique as described above and expressed in terms ofcumulative drug release (%) over time as shown in FIG. 4. In FIG. 4, thein vitro drug release studies (1 ml of liposomes dialysed against 40 mlof PBS with pH of 7.4) for three independent samples of tacrolimusloaded DPPC liposomes as carried out are shown. The cumulativetacrolimus release (%) was plotted against time (days). For each. Therelease of tacrolimus from liposomes was sustained beyond two weeks invitro (about 35%), albeit a smaller burst observed in the first fewdays. In comparison with EggPC liposomes, DPPC liposomes has a smallerinitial burst and only about 35% of the drug is released at the end oftwo weeks compared to EggPC liposomes (about 60%).

While the invention has been particularly shown and described withreference to specific embodiments, it should be understood by thoseskilled in the art that various changes in form and detail may be madetherein without departing from the spirit and scope of the invention asdefined by the appended claims. The scope of the invention is thusindicated by the appended claims and all changes which come within themeaning and range of equivalency of the claims are therefore intended tobe embraced.

1. The use of a nanoliposome in the manufacture of a medicament for theprophylaxis and/or treatment of anterior segment ocular diseases,wherein the nanoliposome comprises a plurality of unsaturated and/orsaturated lipids forming at least one lipid bilayer encapsulating ahydrophobic drug comprising tacrolimus, and wherein the hydrophobic drugand the plurality of unsaturated and/or saturated lipids have a weightratio of up to 0.2.
 2. The use according to claim 1, wherein thenanoliposome has a drug loading of up to 1 mg/ml.
 3. The use accordingto claim 1 or 2, wherein the plurality of unsaturated and/or saturatedlipids are selected from the group consisting of phosphocholines andsphingolipids.
 4. The use according to claim 3, wherein thephosphocholines are selected from the group consisting of egg yolkphosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) anddipalmitoylphosphatidylcholine (DPPC).
 5. The use according to any oneof claims 1 to 3, wherein the plurality of unsaturated and/or saturatedlipids comprise at least one unsaturated lipid constituting more than 50wt % of the at least one lipid bilayer.
 6. The use according to claim 5,wherein the at least one unsaturated lipid comprise egg yolkphosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingolipidsand/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
 7. The useaccording to any one of claims 1 to 6, wherein the nanoliposome has anaverage size of 80 nm to 150 nm before storage at 4° C.
 8. The useaccording to any one of claims 1 to 7, wherein the nanoliposome has anaverage size of 90 nm to 120 nm after storage at 4° C.
 9. The useaccording to any one of claims 1 to 8, wherein the nanoliposome has apolydispersity index of less than 0.3 before and after storage.
 10. Theuse according to any one of claims 1 to 9, wherein the nanoliposome is amultilamellar vesicle or an unilamellar vesicle.
 11. A nanoliposome foruse in the prophylaxis and/or treatment of anterior segment oculardiseases, wherein the nanoliposome comprises a plurality of unsaturatedand/or saturated lipids forming at least one lipid bilayer encapsulatinga hydrophobic drug comprising tacrolimus, and wherein the hydrophobicdrug and the plurality of saturated and/or unsaturated lipids have aweight ratio of up to 0.2.
 12. The nanoliposome according to claim 11,wherein the nanoliposome has a drug loading of up to 1 mg/ml.
 13. Thenanoliposome according to claim 11 or 12, wherein the plurality ofunsaturated and/or saturated lipids are selected from the groupconsisting of phosphocholines and sphingolipids.
 14. The nanoliposomeaccording to claim 13, wherein the phosphocholines are selected from thegroup consisting of egg yolk phosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) anddipalmitoylphosphatidylcholine (DPPC).
 15. The nanoliposome according toany one of claims 11 to 13, wherein the plurality of unsaturated and/orsaturated lipids comprise at least one unsaturated lipid constitutingmore than 50 wt % of the at least one lipid bilayer.
 16. Thenanoliposome according to claim 15, wherein the at least one unsaturatedlipid comprise egg yolk phosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingolipidsand/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
 17. Thenanoliposome according to any one of claims 11 to 16, wherein thenanoliposome has an average size of 80 nm to 150 nm before storage at 4°C.
 18. The nanoliposome according to any one of claims 11 to 17, whereinthe nanoliposome has an average size of 90 nm to 120 nm after storage at4° C.
 19. The nanoliposome according to any one of claims 11 to 18,wherein the nanoliposome has a polydispersity index of less than 0.3before and after storage.
 20. The nanoliposome according to any one ofclaims 11 to 19, wherein the nanoliposome is a multilamellar vesicle oran unilamellar vesicle.
 21. A method of preventing and/or treatinganterior segment ocular diseases by administering a nanoliposomecomprising a plurality of unsaturated and/or saturated lipids forming atleast one lipid bilayer encapsulating a hydrophobic drug comprisingtacrolimus, wherein the hydrophobic drug and the plurality of saturatedand/or unsaturated lipids have a weight ratio of up to 0.2.
 22. Themethod according to claim 21, wherein the nanoliposome is administeredby subconjunctival injection.
 23. The method according to claim 21 or22, wherein the nanoliposome administered has a drug loading of up to 1mg/ml.
 24. The method according to any one of claims 21 to 23, whereinthe plurality of unsaturated and/or saturated lipids of the nanoliposomeadministered are selected from the group consisting of phosphocholinesand sphingolipids.
 25. The method according to claim 24, wherein thephosphocholines are selected from the group consisting of egg yolkphosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) anddipalmitoylphosphatidylcholine (DPPC).
 26. The method according to anyone of claims 21 to 24, wherein the plurality of unsaturated and/orsaturated lipids comprise at least one unsaturated lipid constitutingmore than 50 wt % of the at least one lipid bilayer.
 27. The methodaccording to claim 26, wherein the at least one unsaturated lipidcomprise egg yolk phosphatidylcholine (EggPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingolipidsand/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
 28. The methodaccording to any one of claims 21 to 27, wherein the nanoliposomeadministered has an average size of 80 nm to 150 nm before storage at 4°C.
 29. The method according to any one of claims 21 to 28, wherein thenanoliposome administered has an average size of 90 nm to 120 nm afterstorage at 4° C.
 30. The method according to any one of claims 21 to 29,wherein the nanoliposome administered has a polydispersity index of lessthan 0.3 before and after storage.
 31. The method according to any oneof claims 21 to 30, wherein the nanoliposome administered is amultilamellar vesicle or an unilamellar vesicle.